Gemcitabine-resistant Cell Lines Research Articles

Abstract 1087: CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest survival rates of cancers, with a five-year relative survival of 9%. The significant factor contributing to low survivorship is the intrinsic resistance to chemotherapy. In the present study, we elucidate a targetable pathway that facilitates resistance in these cells. One inflammatory pathway used in cancer progression is CXCR2, which binds chemokines CXCL 1-3 and 5-8. Our group and others have shown an association of CXCR2 with tumor progression, angiogenesis and metastasis in PDAC, and chemotherapy resistance in other cancers. In this study, we test the hypothesis that CXCR2 is integral in PDAC chemotherapy resistance. We generated gemcitabine resistant cell lines (GemR) from the parent cell lines of T3M4 (WT KRAS) and CD18/HPAF (Mutant KRAS) with 40- and 67-fold higher resistance in comparison to the parent cell lines, respectively. Using qRT-PCR and ELISA, we profiled chemokines' expression, CXCL1, 5, and 8, on parent- and GemR-cell lines. We observed a higher basal level expression of CXCL1, 5, and 8 ligands in GemR cells than the parent cell lines. Further treatment with gemcitabine, the parent cell lines had a higher increase in CXCL1 and 8 ligand expression in comparison with GemR cell lines. With further investigation of T3M4, we found a dose-dependent and time-dependent increase for CXCL1 and CXCL8. CXCL5 had a constant high expression with all doses of gemcitabine. With our initial observations of gemcitabine treatment enhancing CXCR2 ligands in PDAC cells, we evaluated the effects of the small molecule CXCR2 antagonist SCH 479833 (SCH 47) in combination with gemcitabine treatment on the parent and GemR cell lines. We used the relative IC50 and half the relative IC50 for both the gemcitabine and SCH 47 treatment doses. We observed that a lower concentration of gemcitabine in the presence of the SCH 47 was more effective than the higher concentration of gemcitabine without SCH 47 for the GemR-cell lines. Together, our observations show chemotherapy-resistant PDAC cell lines express higher CXCR2 ligand levels at baseline, with parent cell lines increasing expression with chemotherapy treatment. We also show an advantageous combination treatment of CXCR2 antagonist with lower gemcitabine concentration. Overall, these results show a promising future for CXCR2 antagonism in the treatment of resistant PDAC tumors. Citation Format: Caitlin Molczyk, Dipakkumar R. Prajapati, Sugandha Saxena, Paran Goel, Michelle Varney, Rakesh K. Singh. CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1087.

Zebularine suppressed gemcitabine-induced senescence and improved the cellular and plasma pharmacokinetics of gemcitabine, augmented by liposomal co-delivery

Chemoresistance is a major factor driving cancer recurrence. This study investigated the potential of zebularine, a dual cytidine deaminase (CDA)/epigenetic inhibitor, to circumvent gemcitabine-resistance in pancreatic cancer using a nanomedicine co-delivery approach. The mRNA expression of key metabolic enzymes, including CDA for gemcitabine deactivation in a gemcitabine-resistant cell line Gr2000 and its parental MIA PaCa-2 was compared using quantitative reverse transcription polymerase chain reaction. A highly gemcitabine-resistant population (HRP) in Gr2000 were characterised for their growth pattern, β-galactosidase activity (a hallmark of senescence) and chemosensitivity to zebularine after isolation. The CDA inhibition effects of zebularine on the intracellular gemcitabine accumulation and pharmacokinetics in rats when co-delivered with pH-sensitive liposomes (pSL) were investigated. Gr2000 had a 3-time upregulated mRNA expression and enzyme activity for CDA. The HRP (28% of bulk Gr2000) were predominately senescent cells which re-proliferated following a growth arrest for a week. Zebularine suppressed the regrowth of senescent cells, meanwhile enhanced cellular gemcitabine concentration by 2-fold. When co-delivered with pSL, zebularine increased cellular gemcitabine concentration by 4-fold, and extended the half-life of gemcitabine in plasma by 22-fold in rats. In conclusion, multiple mechanisms including therapy-induced senescence were identified with gemcitabine-resistance. Co-delivery of zebularine using liposomes could provide multifaceted benefits in gemcitabine therapy for pancreatic cancer treatment.

Effects of Low Dose Fractional Radiation on Chemosensitivity of Gemcitabine-Resistant Human Pancreatic Cancer SW1900/GZ Cell

Objective: To investigate whether low-dose fractionated radiation (LDFRT) could enhance gemcitabine sensitivity in drug-resistant human pancreatic cancer SW1900/GZ cell, and to further explore the underlying mechanism. Methods: Gemcitabine-resistant human pancreatic cancer SW1900 cell line (SW1900/GZ) was induced by high concentration gemcitabine intermittent shock in vitro. The cell counting kit 8 (CCK8) was used to determine SW1900/GZ cell lines. SW1900/GZ cells were divided into six groups as follows: control, LDFRT, high dose radiation (HDRT), gemcitabine (GEM), low dose fractional radiation plus gemcitabine (LDFRT+ GEM) and high dose radiation plus gemcitabine (HDRT+ GEM) groups. The rate of apoptosis was determined by flow cytometry (FCM). Protein levels of multidrug resistance gene (MDR) and multidrug resistance-related protein gene (MRP) were examined by Western blotting. Results: The results of CCK8 test showed that the half-maximal inhibitory concentration (IC50) of non-drug-resistant cell line SW1900 and drug-resistant cell line SW1990/GZ were 230.4ng/ml and 856.6ug/ml respectively. The IC50 of SW1990/GZ was 3700 times more than the former. LDFRT significantly promoted apoptosis in SW1900 cells. Moreover, in the LDFRT group, protein levels of MDR and MRP were markedly decreased. Conclusion: This study established an effective gemcitabine-resistant cell line SW1900 of human pancreatic cancer (SW1900/GZ cell line). LDFRT sensitizes resistant SW1900/GZ pancreatic cancer cell to gemcitabine through down-regulation the expression of MDR and MPR proteins.

Suppression of Wnt/β-catenin and RAS/ERK pathways provides a therapeutic strategy for gemcitabine-resistant pancreatic cancer

Pancreatic cancer is a major malignant tumor without an effective treatment. KRAS mutations occur in 90% of the pancreatic cancer patients and are a major obstacle for treatment of pancreatic cancer. Pancreatic cancer patients have been treated with limited chemotherapeutic agents such as gemcitabine. However, patients often develop resistance to gemcitabine that is attributed to KRAS mutations. Gemcitabine treatment activates both the Wnt/β-catenin and RAS/ERK pathways. These signaling pathways are also activated in the gemcitabine-resistant pancreatic cancer cell lines, suggesting that they play an important role in gemcitabine resistance in pancreatic cancer. The gemcitabine-resistant cell lines show enhanced migratory and invasive capabilities than their parental lines. Therefore, we investigated the effects of a small molecule, KYA1797K that degrades both β-catenin and RAS, on pancreatic cancer. KYA1797K decreased the expression level of both β-catenin and KRAS in pancreatic cancer cell lines expressing either wild-type or mutant KRAS. It also suppressed migration and invasion of gemcitabine-resistant and parental pancreatic cancer cells. Overall, we demonstrated that inhibiting the Wnt/β-catenin and RAS/ERK pathways by destabilizing β-catenin and RAS could be a therapeutic approach to overcome gemcitabine resistance in pancreatic cancer.

MicroRNA-136-5p regulates gemcitabine resistance in pancreatic cancer via down-regulating ZNF32.

We explored the regulation of microRNA-136-5p on gemcitabine resistance of pancreatic cancer (PCa) and further highlighted the crosstalk between microRNA-136-5p and ZNF32, so as to provide an effective theoretical basis for target treatment of PCa. MicroRNA-136-5p and ZNF32 levels in tumor specimens of 48 patients with PCa were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method, and the interplay between microRNA-136-5p and clinicopathological parameters, as well as prognosis of those patients was also analyzed. Meanwhile, in gemcitabine-resistant PCa cell lines PANC-1 and CFPAC-1, microRNA-136-5p overexpression model was constructed, and cell counting kit-8 (CCK-8), transwell, as well as cell wound healing assays were carried out to assess the impact of microRNA-136-5p on PCa cell functions. Finally, Luciferase assay and recovery experiments were conducted to specify the precise underlying mechanism. qRT-PCR results revealed a significant low expression of microRNA-136-5p both in tumor tissues of PCa patients and in PCa cell lines compared to the normal control groups. In addition, microRNA-136-5p mimics markedly attenuated the proliferation and migration abilities of the gemcitabine-resistant PCa cell lines. The Luciferase assay verified certain binding sites between microRNA-136-5p and ZNF32, while qPCR results indicated a negative correlation between the two in PCa tissues. Moreover, recovery experiments demonstrated that ZNF32 overexpression reversed the inhibitory effect on the malignant progression of gemcitabine-resistant PCa cells induced by microRNA-136-5p. MicroRNA-136-5p can reduce the proliferation rate and metastasis ability of PCa cells via regulating ZNF32, and thus alleviates gemcitabine resistance in PCa cells.

In silico identification of therapeutic compounds against microRNA targets in drug-resistant pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a major health issue that has been eluding efforts to identify viable therapeutic treatment options. Besides having the lowest survival rate among all types of cancer, almost all conventional methods of treatment are futile against this condition, leaving patients to succumb to this ailment faster than ever. As it is increasingly becoming difficult to come up with new compounds for the treatment of various diseases, alternative solutions are required for tackling these problems. In this study, publically available miRNA and gene expression data were used to identify common elements that were present in gemcitabine-resistant PDAC cell lines. By selecting overexpressed genes involved in pancreatic cancer and cancer pathways in general, potential drug candidates for the treatment of PDAC were identified. In this study, 21 differentially expressed miRNAs were identified from PANC-1 cell line treated with gemcitabine. Pathway analysis revealed that MET and PPARG were overexpressed in cancer-related pathways, including pancreatic cancer, and could be targeted for PDAC treatment. Using CMap, fisetin was identified a likely candidate drug for the treatment of PDAC. Docking studies indicated that fisetin was bound to c-Met and PPARG with an XP G score of –12.819 and –7.021 kcal/mol, respectively. As miRNAs have increasingly been shown to part take in important cancer-related processes and pathways, researching drug development methods based on miRNA targets could be beneficial for pharmaceutical industries. Communicated by Ramaswamy H. Sarma

PCL20-117: Can Green Tea Extract Inhibit the Tumor Growth and Invasiveness of 5-FU and Gemcitabine-Resistant Metastatic and Remetastatic Cell Lines in a Hamster Pancreatic Cancer Model?

PCL20-117: Can Green Tea Extract Inhibit the Tumor Growth and Invasiveness of 5-FU and Gemcitabine-Resistant Metastatic and Remetastatic Cell Lines in a Hamster Pancreatic Cancer Model?

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

SummaryIn situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and β-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

Activation of GLP-1 receptor enhances the chemosensitivity of pancreatic cancer cells.

This study aimed to determine whether and how the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide affects the chemoresistance and chemosensitivity of pancreatic cancer cells to gemcitabine in vitro and in vivo. The GLP-1R and protein kinase A (PKA) levels were compared between the human pancreatic cancer cell line PANC-1 and the gemcitabine-resistant cell line PANC-GR. The in vitro effects of liraglutide on the cell proliferation and apoptosis as well as the nuclear factor-kappa B NF-κB expression levels of PANC-GR cells were evaluated. In addition, a mouse xenograft model of human pancreatic cancer was established by s.c. injection of PANC-1 cells, and the effects of liraglutide on the chemosensitivity were evaluated in vitro and in vivo. In contrast to PANC-1 cells, PANC-GR cells exhibited lower expression levels of GLP-1R and PKA. Incubation with liraglutide dose dependently inhibited the growth, promoted the apoptosis, and increased the expression of GLP-1R and PKA of PANC-GR cells. Similar effects of liraglutide were observed in another human pancreatic cancer cell line MiaPaCa-2/MiaPaCa-2-GR. Either the GLP-1R antagonist Ex-9, the PKA inhibitor H89, or the NF-κB activator lipopolysaccharide (LPS) could abolish the antiproliferative and proapoptotic activities of liraglutide. Additionally, each of these agents could reverse the expression of NF-κB and ABCG2, which was decreased by liraglutide treatment. Furthermore, liraglutide treatment increased the chemosensitivity of pancreatic cancer cells to gemcitabine, as evidenced by in vitro and in vivo experiments. Thus, GLP-1R agonists are safe and beneficial for patients complicated with pancreatic cancer and diabetes, especially for gemcitabine-resistant pancreatic cancer.

LncRNA SLC7A11-AS1 Promotes Chemoresistance by Blocking SCFβ-TRCP-Mediated Degradation of NRF2 in Pancreatic Cancer

Drug resistance is the major obstacle of gemcitabine-based chemotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC). Many long non-coding RNAs (lncRNAs) are reported to play vital roles in cancer initiation and progression. Here, we report that lncRNA SLC7A11-AS1 is involved in gemcitabine resistance of PDAC. SLC7A11-AS1 is overexpressed in PDAC tissues and gemcitabine-resistant cell lines. Knockdown of SLC7A11-AS1 weakens the PDAC stemness and potentiates the sensitivity of resistant PDAC cells toward gemcitabine in vitro and in vivo. SLC7A11-AS1 promotes chemoresistance through reducing intracellular reactive oxygen species (ROS) by stabilizing nuclear factor erythroid-2-related factor 2 (NRF2), the key regulator in antioxidant defense. Mechanically, SLC7A11-AS1 is co-localized with β-TRCP1 in the nucleus. The exon 3 of SLC7A11-AS1 interacts with the F-box motif of β-TRCP1, the critical domain that recruits β-TRCP1 to the SCFβ-TRCP E3 complex. This interaction prevents the consequent ubiquitination and proteasomal degradation of NRF2 in the nucleus. Our results demonstrate that the overexpression of SLC7A11-AS1 in gemcitabine-resistant PDAC cells can scavenge ROS by blocking SCFβ-TRCP-mediated ubiquitination and degradation of NRF2, leading to a low level of intracellular ROS, which is required for the maintenance of cancer stemness. These findings suggest SLC7A11-AS1 as a therapeutic target to overcome gemcitabine resistance for PDAC treatment.

Long non-coding RNA SSTR5-AS1 facilitates gemcitabine resistance via stabilizing NONO in gallbladder carcinoma

Gallbladder carcinoma (GBC) is the most aggressive carcinoma of the biliary tract, effective chemotherapy was critical for the patients with unresectable GBC. However, chemotherapy resistance is still problematic for clinicians. Here, we identified a specific long non-coding RNA, SSTR5-AS1, in GBC patient that facilitates gemcitabine resistance. SSTR5-AS1 is significantly increased in GBC samples and cell lines, especially in gemcitabine-resistant cell lines, and higher SSTR5-AS1 expression was correlated with poorer overall survival rate in GBC patients. Our data revealed that upregulated SSTR5-AS1 facilitates gemcitabine resistance via inhibiting apoptosis. Knockdown of SSTR5-AS1 sensitized drug resistant GBC cells to gemcitabine in vitro and strongly inhibited xenografts formed by drug resistant GBC cells in vivo. Moreover, we found via streptavidin pull down assay that NONO specifically binds to sense sequence of SSTR5-AS1 and prevented proteasome mediated NONO degradation, which resulted in increased NONO protein level without affecting the transcription of NONO. NONO functions as the downstream effector of SSTR5-AS1 and is required for SSTR5-AS1 mediated gemcitabine resistance. Collectively, our data provided novel insights into lncRNA-mediated chemotherapy resistance and suggested a novel therapeutic target to improve chemotherapy strategies for unresectable GBC patients.

Anti-parasitic Drug Ivermectin Exhibits Potent Anticancer Activity Against Gemcitabine-resistant Cholangiocarcinoma In Vitro

The antiparasitic drug, ivermectin (IVM), exerts anticancer activities in diverse cancer types. However, its anticancer activity against cholangiocarcinoma (CCA), especially the drug-resistant phenotype, has not yet been explored. IVM was tested for its anticancer activity against gemcitabine-sensitive (KKU214) and gemcitabine-resistant (KKU214GemR) CCA cell lines in vitro using the sulforhodamine B and clonogenic assays as well as cell-cycle analysis. IVM treatment inhibited cell proliferation and colony formation of both KKU214 and KKU214GemR in a dose- and time-dependent manner. KKU214GemR cells were more sensitive than KKU214 to IVM treatment. IVM treatment caused S-phase cell-cycle arrest and also cell death as indicated by an increase of sub-G0/G1 population in KKU214GemR cells treated with IVM for 48 h. IVM exerts anti-CCA activities and gemcitabine-resistant KKU214GemR cells are more sensitive to IVM treatment. Thus, IVM might be useful as an alternative treatment for CCA, especially in patients who do not respond to gemcitabine.

Screening miRNAs associated with resistance gemcitabine from exosomes in A549 lung cancer cells.

PurposeTo establish a gemcitabine-resistant lung adenocarcinoma cell line, A549/G+, and to screen the differences of miRNA expression in exosomes from A549 and A549/G+ cells.MethodsA549 cells were exposed in gemcitabine until they were resistant to gemcitabine, and extracted exosomes from A549 and A549/G+. The RNAs from exosomes were subjected to miRNA expression microarray experiments.ResultsAfter 39 weeks of continuous induction, we induced drug resistance in A549 cells. The resistance index was 6. Via GeneChip miRNA 4.0 analysis, there were 446 differential miRNAs between A549 and A549/G+. Target gene prediction and pathway analysis discovered the microRNAs in the intersections may participate in drug resistance.ConclusionThese differential miRNAs help to do in-depth research to elucidate the mechanism of resistance to gemcitabine in non-small cell lung cancer.

Abstract 3021: The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis and an increased chemoresistance. Dysfunction in alternative splicing has been associated with chemoresistance in many cancers. Alternative splicing of survivin mRNA generates six splicing isoforms some of which are associated with cancer progression and chemoresistance. Survivin is a member of the inhibitors of apoptosis (IAPs) family and a cell cycle-associated oncoprotein overexpressed in almost all cancers. Interestingly, some of these survivin splice variants are proapoptotic (2α and 2β) while others are antiapoptotic (survivin, Δex3, 3α and 3β). It has been reported that increased levels of survivin 2β and 3β play a role in chemoresistance in ovarian cancers and other tumors, but this has not been studied in pancreatic cancer. Our recent RT-qPCR data also show an overexpression of all survivin splice variants in chemoresistant PDAC cell lines compared to the chemo-sensitive cell line. We, therefore, hypothesize that increased expression of survivin splice variants 2β and 3β contribute to chemo-resistance in PDAC. To test this hypothesis, we repeatedly selected the resistant cells from the normally chemosensitive MiaPaca2 cell line after exposure to gemcitabine. We then assessed the levels of survivin splice variants in the new cell line (gemcitabine-resistant MiaPaca2). Using the Alamar blue assay, we confirmed that Panc1 and gemcitabine-resistant MiaPaca2 are more resistant to gemcitabine compared to MiaPaca2 cells. We also observed a statistically significant overexpression of survivin 2β and 3β in gemcitabine-resistant cell lines compared to the more sensitive MiaPaca2 cells. To further test our hypothesis, we will knockdown survivin 2β and 3β in Panc1 and gemcitabine-resistant MiaPaca2, and/or overexpress these splice variants in MiaPaca2 cells then assess response to gemcitabine. The outcomes of this study might create new biomarkers for predicting the clinical outcomes of gemcitabine treatment and generate new therapeutic targets for pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Janviere Kabagwira, Amber Gonda, Mei Li Kwong. The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3021.

Long non-coding RNA GHET1 contributes to chemotherapeutic resistance to Gemcitabine in bladder cancer.

Bladder cancer (BC) ranks first in the incidence of urogenital tumors in China and second only to prostate cancer in the West. This study will clarify the roles and mechanism of lncRNA GHET1 in chemotherapeutic resistance of BC to Gemcitabine. The expression of GHET1 was examined using real-time quantitative PCR. Cell Counting Kit-8 assay was applied to analyze cell proliferation and Gemcitabine sensitivity. Cell apoptosis was detected using Annexin V-FITC/PI double-stained flow cytometry. The expression of ABCC1 protein was examined using Western blotting. Firstly, the expression of GHET1 was up-regulated in BC, its high expression was relevant to high grade and muscle invasion of BC patients. Secondly, high expression of GHET1 was related to low Gemcitabine sensitivity of BC patients, and GHET1 was highly expressed in Gemcitabine-resistant BC cell lines. Thirdly, knockdown of GHET1 decreased the IC50 of Gemcitabine in Gemcitabine-resistant BC cell lines and advanced the Gemcitabine-induced cytotoxicity; GHET1 promoted Gemcitabine resistance in BC. Finally, knockdown of GHET1 also inhibited the expression of ABCC1 protein in Gemcitabine-resistant BC cells. High expression of GHET1 was related with the low sensitivity to Gemcitabine of BC; GHET1 contributed to chemotherapeutic resistance to Gemcitabine in BC through up-regulating ABCC1 expression. Our findings are helpful to expound the molecular mechanism of chemotherapeutic resistance in BC.

Recombinant deoxyribonucleoside kinase from Drosophila melanogaster can improve gemcitabine based combined gene/chemotherapy for targeting cancer cells.

A recombinant deoxyribonucleoside kinase from Drosophila melanogaster with a deletion of the last 20 amino acid residues (named DmdNKΔC20) was hypothesized as a potential therapeutic tool for gene therapy due to its broad substrate specificity and better catalytic efficiency towards nucleosides and nucleoside analogs. This study was designed to evaluate the effect of DmdNKΔC20 for sensitizing human cancer cell lines to gemcitabine and to further investigate its role in reversal of acquired drug resistance in gemcitabine-resistant cancer cell line. The DmdNKΔC20 gene was delivered to three different cancer cell lines, including breast, colon and liver cancer cells, using lipid-mediated transfection reagent. After transfection, gene expression of DmdNKΔC20 was confirmed by quantitative reverse transcription PCR (qRT-PCR) and the combined effect of DmdNKΔC20 and gemcitabine based cytotoxicity was observed by cell viability assay. We further evolved a gemcitabine-resistant breast cancer cell line (named MCF7-R) through directed evolution in the laboratory, which showed 375-fold more resistance compared with parental MCF7 cells. Upon transfection with DmdNKΔC20 gene, MCF7-R cells showed 83-fold higher sensitivity to gemcitabine compared with the control group of MCF7-R cells. Moreover, we observed 79% higher expression of p21 protein in transfected MCF7-R cells, which may indicate induction of apoptosis. Our findings highlight the importance and therapeutic potential of DmdNKΔC20 in combined gene/chemotherapy approach to target a wide range of cancers, particularly gemcitabine-resistant cancers.

Antitumor effects of all-trans retinoic acid and its synergism with gemcitabine are associated with downregulation of p21-activated kinases in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide. All-trans retinoic acid (ATRA) has been used as an antistromal agent in PDA, and its antitumor effect has also been reported in various kinds of cancer, including PDA. Inhibition of p21-activated kinases (PAKs) is associated with decreased tumor growth and increased gemcitabine sensitivity. The aim of this study was to evaluate the inhibitory effects of ATRA alone and in combination with gemcitabine on cell growth and migration of wild-type and gemcitabine-resistant PDA cells and the potential mechanism(s) involved. Human (MiaPaCa-2) and murine (TB33117) PDA cell lines were incubated in increasing concentrations of gemcitabine to establish resistant clones. Cell growth, clonogenicity, and migration/invasion were determined using a sulforhodamine B assay, a colony formation assay, and a Boyden chamber assay, respectively. Protein expression was measured by Western blotting. ATRA reduced cell proliferation, colony formation, and migration/invasion in both wild-type and gemcitabine-resistant cell lines. PAK1 expression was significantly increased in resistant cells. Cells treated with ATRA showed decreased expression of PAK1, PAK2, PAK4, and α-smooth muscle actin. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant cell lines. Depletion of PAK1 enhanced ATRA sensitivity in MiaPaCa-2 cells. In conclusion, the antitumor effects of ATRA and its synergism with gemcitabine are associated with downregulation of PAKs. NEW & NOTEWORTHY The inhibitory effect of all-trans retinoic acid (ATRA) on cell proliferation, colony formation, and migration/invasion was associated with downregulation of p21-activated kinases (PAKs), and depletion of PAK1 enhanced ATRA sensitivity in MiaPaCa-2 cells. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant pancreatic ductal adenocarcinoma cells. As an important prognostic marker, α-smooth muscle actin also can be downregulated by ATRA in pancreatic ductal adenocarcinoma cells.

Enhanced expression of ten-eleven translocation 1 reverses gemcitabine resistance in cholangiocarcinoma accompanied by a reduction in P-glycoprotein expression.

Increasing evidence revealed that ten‐eleven translocation 1 (TET1) plays an important role in tumorigenesis and chemoresistance, but its functions in gemcitabine resistance in cholangiocarcinoma (CCA) remain unknown. This study aims to investigate the effect of TET1 on gemcitabine resistance in CCA and the possible effect on P‐glycoprotein (P‐gp) expression encoded by multidrug resistance (MDR) genes. We established two kinds of gemcitabine‐resistant CCA cell lines and confirmed its specific features. The expression of TET1 and P‐gp was evaluated in gemcitabine‐resistant CCA cells and their parental cells at mRNA and protein level by quantitative RT‐PCR and western blot analysis. After transfecting the gemcitabine‐resistant CCA cell lines with TET1 gene or siRNA, the cell viability test was obtained to verify the effect of TET1 on the sensitivity of CCA cells to gemcitabine. And then, the possible effect of TET1 on the expression of P‐gp was examined by western blot analysis. Xenograft tumor experiment was conducted to confirm the association between TET1 and P‐gp expression under gemcitabine chemoresistance. The associations between clinical outcomes of CCA patients with chemotherapy and TET1 expression were analyzed in 82 patients. The results showed that TET1 expression was significantly decreased, and P‐gp expression was increased in gemcitabine‐resistant CCA cells. Additionally, overexpression of TET1 augmented the sensitivity of CCA cells to gemcitabine and induced the decreased expression of P‐gp in gemcitabine‐resistant CCA cells. Furthermore, multivariate Cox regression analysis indicated that TET1 expression and TNM stage were independent risk factors (P < 0.001) for the clinical outcomes of CCA patients with chemotherapy. Additionally, Kaplan‐Meier survival and the log‐rank test showed that decreased expression of TET1 was associated with poorer prognosis of CCA patients with chemotherapy. These findings suggest that TET1 expression reverses gemcitabine resistance in CCA accompanied by a reduction in P‐gp expression. Thus, TET1 may be a promising target to overcome chemoresistance in CCA.

Comprehensive analysis of microRNA-messenger RNA regulatory network in gemcitabine-resistant bladder cancer cells.

Chemotherapy is still a standard treatment of unresectable bladder cancer or distant metastases. The chemotherapy resistance always occurs after a period of treatment indicating poor prognosis. The current study aimed to explore the molecular mechanism of chemoresistance in bladder cancer cells. The gene expression profiles of GSE77883, including three untreated T24 cells samples and three gemcitabine-resistant T24 cells samples, was downloaded from Gene Expression Omnibus database. The screening of differentially expressed genes (DEGs), gene function analysis, and interaction prediction between microRNAs (miRNAs) and DEGs were performed by R software. The protein-protein interaction (PPI) and miRNA-DEGs networks were constructed and visualized by Cytoscape software. Then, the small molecules, with potential synergistic or antagonistic effects to gemcitabine resistance, were identified using the Connectivity Mapdatabase. Finally, gemcitabine-resistant T24 cell line was established and key genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR). In total, 536 upregulated and 513 downregulated genes were screened and mainly enriched in oxidative stress response and signaling pathways related to extracellular matrix-receptor interaction and focal adhesion. PPI network showed interleukin 6, tumor necrosis factor, kinesin family member 11, and BUB1 mitotic checkpoint serine/threonine kinase B were key genes. The miRNA-DEGs regulatory networks included 18 miRNAs and 185 DEGs, including miR-182-5p, miR-590-3p, miR-320a and serum- and glucocorticoid-regulated kinase 1 (SGK1). Then, the related key genes and miRNAs were confirmed by qRT-PCR. Furthermore, 81 small molecules with antagonistic or synergistic effect to GEM were screened. We have investigated the molecular mechanisms driving GEM-resistance in bladder cancer cells that would contribute to the development of chemotherapy for advanced bladder cancer.

Hypoxia-Induced PLOD2 is a Key Regulator in Epithelial-Mesenchymal Transition and Chemoresistance in Biliary Tract Cancer.

The prognosis of biliary tract cancer (BTC) is unfavorable due to its chemoresistance. Hypoxia triggers epithelial-to-mesenchymal transition (EMT), which is closely related to drug resistance. In this study, we focused on the functional roles of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a hypoxia-induced gene, in BTC, and assessed the clinical significance of PLOD2. The expression of PLOD2 under hypoxia was assessed in BTC cell lines. Gemcitabine-resistant (GR) BTC cell lines were transfected with small interfering RNA (siRNA) against PLOD2, and EMT markers and chemoresistance were evaluated. PLOD2 expression was also characterized using immunohistochemistry in BTC clinical specimens following resection. Patient survival was analyzed and the role of PLOD2 expression was examined. The expression of PLOD2 was induced by hypoxia in vitro and was upregulated in BTC-GR cell lines, which had low expression of epithelial markers and high expression of mesenchymal markers. Downregulation of PLOD2 by siRNA resulted in improved chemoresistance, recovery of epithelial markers, and reduction of mesenchymal markers. In the resected BTC samples, PLOD2 expression was significantly correlated with lymph node metastasis (p = 0.037) and stage (p = 0.001). Recurrence-free survival (p = 0.011) and overall survival (p < 0.001) rates were significantly lower in patients with high expression of PLOD2. PLOD2 expression was an independent prognostic factor for overall survival (p = 0.019). The expression of PLOD2 influenced chemoresistance through EMT, and high expression of PLOD2 was a significant unfavorable prognostic factor in BTC patients. PLOD2 might be a potential therapeutic target for overcoming chemoresistance.